Arterial calcification is a highly regulated form of biomineralization that proceeds via mechanisms resembling membranous and endochondral bone formation. Reactive oxygen species (ROS), arising from the low grade inflammation of diabetes and dyslipidemia, initiate osteogenic signals that promote valve and arterial calcium accrual. Msx2 -- Wnt cascades are recruited to direct osteogenic lineage allocation of vascular myofibroblasts; this is amplified by osteochondrogenic trans-differentiation of vascular smooth muscle cells (VSMCs) - a characteristic change with hyperphosphatemia and atherosclerosis. In patients with type II diabetes (T2DM), medial artery calcification is a powerful index of lower extremity amputation risk; arteriosclerotic medial calcification is a much more ominous predictor of amputation than atherosclerotic calcification. Strategies that reduce aortofemoral arteriosclerosis and maintain vascular compliance will improve tissue perfusion, and mitigate amputation risk. Previously, we examined the impact of inflammation, teriparatide (suppresses vascular Msx2), and osteopontin (OPN) on aortic structure, calcification, and compliance. OPN actions emerged as biologically complex and stage-specific. Diabetic LDLR-/- mice null for OPN exhibit reduced aortic macrophage content, inflammation, and ROS - yet exhibit increased aortic stiffness even without calcification. Although initially reduced, aortic calcium load subsequently becomes more severe in diabetic OPN-null mice with disease progression. A protease-liberated OPN fragment promotes vascular ROS and MMP9 activation. Thus, OPN is an inflammatory pro-hormone as well as a mineralization inhibitor and adhesion molecule. A better understanding of OPN and Msx2 in the biology of diabetic arterial calcification is needed. Therefore, specific aims are: Aim 1: To define the roles of VSMC vs. macrophage OPN in regulation of diabetic aortic calcification and compliance, using SM22- OPNTg(+);OPN-/-;LDLR-/- and SRA-OPNTg(+);OPN-/-;LDLR-/- mice as models for study. We study OPN actions in VSMC (SM22) and the monocyte/macrophage lineage (scavenger receptor A promoter), evaluating the impact on mural inflammation, macrophage accumulation, matrix remodeling, aortic structure, arterial calcium accrual, and aortic compliance. Aim 2: To establish the role of vascular Msx2 on diabetic aortic calcification, evaluating diabetic SM22- CreTg(+);Msx2(flox/flox);LDLR-/- mice as a model for study. The identification of distinct phases of initiation vs. progression in diabetic arterial calcification has significant implications for vascular health management. Thus, we evaluate the roles of vascular Msx2 and OPN on initiation vs. progression phases of diabetic arteriosclerotic disease. PUBLIC HEALTH RELEVANCE: Arterial hardening in diabetes increases the risk for heart attacks & foot amputation. This occurs in part via metabolic & inflammatory signals that induce bone-like calcification in large arteries. We test whether regulation of these signals reduces arterial hardening, decreases cardiac workload, and improves blood flow to the foot.